To facilitate the international food trade as well as to protect consumers from exposure to unacceptable pesticide residue levels, Codex Alimentarius Commission, European Union, and National Authorities set maximum residue limits for different food commodities. The control of pesticide residues at national and international level requires reliable and comparable analytical data that can be obtained by applying validated methods and implementing an effective internal quality control and quality assurance system in the testing laboratories. For the correct interpretation of the analytical results, measurement uncertainty should be estimated. Pesticide residue analysis includes two main steps: sampling performed outside of the laboratory and laboratory operations comprising of sample preparation, sample size reduction, sample processing, extraction, cleanup, and chromatographic determination. By taking into consideration the contribution of the individual steps to the overall uncertainty of the results, the analytical procedures can be optimized to fit for the purpose of the analysis with minimum cost. The scope of this paper is to review major steps of pesticide residue analysis in the light of current developments, to highlight the importance of identification and estimation of the uncertainties associated with the results, to describe suitable methods for their estimation, and to summarize the contribution of each step to the combined uncertainty.